U.S. patent application number 11/519454 was filed with the patent office on 2007-05-24 for mycoplasma detection method and composition.
Invention is credited to Michael G. Anderson, Jackie A. Ernst, Kim M. Herman-Hatten, James J. Rivard, Paul Younge.
Application Number | 20070117120 11/519454 |
Document ID | / |
Family ID | 37865519 |
Filed Date | 2007-05-24 |
United States Patent
Application |
20070117120 |
Kind Code |
A1 |
Anderson; Michael G. ; et
al. |
May 24, 2007 |
Mycoplasma detection method and composition
Abstract
A method of detecting the presence of mycoplasma in a sample and
a mycoplasm detection kit. A sample is contacted with a first and a
second oligonucleotide probe, wherein the first and second
oligonucleotide probes are substantially complementary to portions
of a 16S ribosomal subunit of at least one mycoplasma species. The
first probe may be labeled with a capture ligand. The second probe
may be labeled with a first component of a detection system.
Inventors: |
Anderson; Michael G.;
(Maplewood, MN) ; Ernst; Jackie A.; (Elk River,
MN) ; Herman-Hatten; Kim M.; (Champlin, MN) ;
Rivard; James J.; (Albertville, MN) ; Younge;
Paul; (Minneapolis, MN) |
Correspondence
Address: |
INTELLECTUAL PROPERTY GROUP;FREDRIKSON & BYRON, P.A.
200 SOUTH SIXTH STREET
SUITE 4000
MINNEAPOLIS
MN
55402
US
|
Family ID: |
37865519 |
Appl. No.: |
11/519454 |
Filed: |
September 12, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60716326 |
Sep 12, 2005 |
|
|
|
Current U.S.
Class: |
435/6.12 ;
435/6.15 |
Current CPC
Class: |
C12Q 1/689 20130101 |
Class at
Publication: |
435/006 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Claims
1. A method for detecting the presence of mycoplasma in a sample
comprising: a) contacting a sample treated to release ribosomal RNA
of mycoplasma present in the sample with a first and second
oligonucleotide probe wherein the first oligonucleotide probe is
substantially complementary to a portion of a 16S ribosomal RNA of
at least one mycoplasma species and is labeled with a capture
ligand and wherein the second oligonucleotide probe is
substantially complementary to a different portion of the 16S
ribosomal RNA of the at least one mycoplasma species; b) incubating
the sample with the first and second oligonucleotide probes under
hybridization conditions to form a hybridization solution and for a
time sufficient for the probes to hybridize to 16S ribosomal RNA of
mycoplasma species present in the sample; c) contacting the
hybridization solution with a solid phase coated with a capture
receptor capable of specifically binding to the capture ligand of
the first labeled oligonucleotide probe; and d) detecting the
presence of mycoplasma ribosomal RNA in the sample.
2. The method of claim 1 wherein the first oligonucleotide probe is
selected from the group consisting of SEQ ID NOs: 12 to 15 and the
second oligonucleotide probe is selected from the group consisting
of SEQ ID NOs: 1 to 11.
3. The method of claim 2 wherein the capture ligand is biotin.
4. The method of claim 3 wherein the capture receptor on the solid
phase is streptavidin or anti-biotin.
5. The method of claim 1 wherein the second oligonucleotide probe
is labeled with a first component of a detection system wherein the
component comprises a detection ligand.
6. The method of claim 5 wherein the presence of mycoplasma
ribosomal RNA in the sample is detected by detecting the presence
of the detection ligand on the second oligonucleotide probe bound
to mycoplasma ribosomal RNA present in the sample.
7. The method of claim 6 wherein the first oligonucleotide probe is
selected from the group consisting of SEQ ID NOs: 12 to 15 and the
second oligonucleotide probe is selected from the group consisting
of SEQ ID NOs: 1 to 11.
8. The method of claim 6 wherein the detection ligand is detected
by adding another component of the detection system comprising a
detection receptor labeled with a signal generating moiety wherein
the detection receptor is chosen to specifically bind to the
detection ligand and reacting the signal generating moiety with a
substrate solution to produce a detectable signal.
9. The method of claim 8 wherein the detection ligand is digoxigen
and the detection receptor is anti-digoxigenin antibody.
10. The method of claim 2 wherein the sample is contacted with two
or more different first oligonucleotide probes and two or more
different second oligonucleotide probes and wherein the presence of
two or more species of mycoplasma in the sample is detected.
11. The method of claim 10 wherein the species of mycoplasma
detected are selected from the group consisting of Mycoplasma
arginini, Mycoplasma orale; Mycoplasma fermentans; Acholeplasma
laidlawii; Mycoplasma hyorhinis; Mycoplasma pirum; Mycoplasma
hominis; and Mycoplasma salivarium.
12. The method of claim 8 wherein the signal generating moiety is
alkaline phosphatase.
13. The method of claim 12 wherein the detectable signal produced
is colorimetric.
14. The method of claim 12 wherein an amplifying solution is added
that amplifies the detectable signal.
15. The method of claim 1 wherein the first and second
oligonucleotide probes are each substantially complementary to a
portion of 16S ribosomal RNA of the mycoplasma species.
16. The method of claim 1 comprising contacting the sample with one
of each of the first oligonucleotide probes of SEQ ID NOs. 12-15
and one of each of the second oligonucleotide probes of SEQ ID NOs.
1-11.
17. A mycoplasma detection kit for detecting the presence of
mycoplasma contamination comprising: a) two or more different first
oligonucleotide probes wherein each first oligonucleotide probe is
substantially complementary to a portion of a 16S ribosomal RNA of
a mycoplasma species and is labeled with a capture ligand; b) two
or more different second oligonucleotide probes wherein each second
oligonucleotide probe is substantially complementary to a different
portion of the 16S ribosomal RNA of the mycoplasma species than any
of the first oligonucleotide probes and each is labeled with a
first component of a detection system, wherein the first component
comprises a detection ligand; c) a solid phase coated with a
capture receptor chosen to specifically bind to the capture ligand
on the first probes; and d) a detection solution comprising a
second component of the detection system, wherein the second
component comprises a detection receptor labeled with a signal
generating moiety.
18. The mycoplasma detection kit of claim 17 wherein the two or
more first oligonucleotide probes are selected from the group
consisting of SEQ. NOs.: 12 to 15 and wherein the two or more
second oligonucleotide probes are selected from the group
consisting of SEQ. NOs.: 1 to 11.
19. The mycoplasma detection kit of claim 18 comprising each of the
first oligonucleotide probes of SEQ ID NOs. 12 to 15 and each of
the second oligonucleotide probes of SEQ ID NOs. 1-11.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims priority to U.S. application
Ser. No. 60/716,326, filed Sep. 12, 2005, the disclosure of which
is incorporated herein by reference.
FIELD OF THE INVENTION
[0002] This invention relates to the methods and kits for the
detection of Mycoplasma contamination in cell cultures or other
samples.
BACKGROUND OF THE INVENTION
[0003] Mycoplasma is a term used to denote a species included in
the class Mollicutes. They are the smallest and simplest
free-living parasitic organisms known. Mycoplasma are parasites of
many animal species and typically exhibit host and tissue
specificity. In humans, Mycoplasma (M.) pneumoniae is the
respiratory pathogen responsible for atypical pneumonia. Mycoplasma
are frequently isolated from patients with immunodeficiencies
associated with disease states.
[0004] Mycoplasma are common contaminants of eukaryotic cell
cultures and are known to alter the phenotypic characteristics of
host cell lines. The published incidence of mycoplasma infected
cell cultures has ranged from 4 to 92%. Of the 18 most common
species recognized as culture contaminants, M. orale, M. hyorhinis,
M. arginini, M. fermentans, and Acholeplasma (A.) laidlawii are the
most frequently isolated representing 80 to 90% of all isolates.
The small size of mycoplasma allows them to pass through the
commonly used 0.45 .mu.m sterilization filters and mycoplasma are
typically resistant to antibiotics such as penicillin and
streptomycin. Mycoplasma contamination usually does not produce
visible changes in cell culture medium despite the fact it can
reach titers of 10.sup.8 per milliliter. Sources of mycoplasma
contamination include laboratory personnel, reagents, and
mycoplasma contaminated cell lines.
[0005] Mycoplasma contamination is detected by a number of methods.
Microbial culture is generally considered the most sensitive method
for mycoplasma screening and is commonly used as a reference for
the evaluation of any new mycoplasma detection techniques. However,
culturing mycoplasma can take two to four weeks, and cannot detect
fastidious mycoplasma. Moreover, culturing mycoplasma requires
special growth conditions and is generally restricted to
specialized laboratories.
[0006] Another method involves microscopic visualization of
mycoplasma attached to host cells using fluorescent DNA staining
that displays extra-cellular fluorescence as well as extra-nuclear
fluorescence. This method is efficient for mycoplasma screening and
particularly for detection of M. hyorhinis strains that cannot be
cultivated on microbiological media. However, fluorescent staining
cannot detect plasma species that cyto-absorb poorly. Moreover,
this method requires expertise for accurate interpretation of
results.
[0007] Enzyme-linked immunosorbent assays measuring mycoplasma
specific cell-surface antigens have been described but typically
lack sensitivity. A number of polymerase chain reaction (PCR) based
methods have also been described for the detection of mycoplasma
that achieve high sensitivity and may be amenable to species
identification. However, PCR-based detection of mycoplasma is prone
to false positives due to amplicon contamination and false negative
results due to use of excess sample. Mycoplasma screening methods
utilizing select biochemical activity have also been used, but give
inconsistent results when comparing different cell lines.
[0008] While these known methods have provided researchers with
various techniques to determine mycoplasma contamination, these
techniques have several disadvantages as described above. More
efficient, rapid and sensitive methods of detecting contamination
are desired.
SUMMARY OF THE INVENTION
[0009] The present invention comprises an assay method and kit
designed for routine screening of mycoplasma contamination of
cultured cells and other samples. In one embodiment, the method and
kit of the invention detect mycoplasma 16S ribosomal RNA present in
a sample using a calorimetric amplification system with a
sensitivity comparable to PCR. In one aspect of the invention, the
method of the invention is used to detect the presence of at least
one of the most common mycoplasma species found as contaminants in
samples such as cell cultures. Those species include M. hyorhinus,
M. arginini, M. fermentans, M. orale, M. pirum, M. hominis, M.
salivarium, and A. laidlawii. These species account for
approximately 95% of all mycoplasma contaminations.
[0010] In one embodiment, the invention comprises a method for
detecting the presence of mycoplasma in a sample comprising
contacting a sample treated to release ribosomal RNA of mycoplasma
present in the sample with a first and second oligonucleotide probe
wherein the first oligonucleotide probe is substantially
complementary to a portion of a 16S ribosomal RNA of at least one
mycoplasma species and is labeled with a capture ligand and wherein
the second oligonucleotide probe is substantially complementary to
a different portion of the 16S ribosomal RNA of the mycoplasma
species; incubating the sample with the first and second
oligonucleotide probes under hybridization conditions to form a
hybridization solution and for a time sufficient for the probes to
hybridize to 16S ribosomal RNA of mycoplasma species present in the
sample; and contacting the hybridization solution with a solid
phase coated with a capture receptor capable of specifically
binding to the capture ligand of the first labeled oligonucleotide
probe and detecting the presence of mycoplasma ribosomal RNA in the
sample.
[0011] In one embodiment, the first oligonucleotide probe is one of
the following capture probes: SEQ ID NO. 12--ggataacgct tgcaacctat
gtattaccg; SEQ ID NO. 13--ggtgtgtaca agacccgaga acgtattcac; SEQ ID
NO. 14--ggtgtgtaca aaacccgaga acgtattcac; and SEQ ID NO.
15--ggtgtgtaca aaccccgaga acgtattcac and the second oligonucleotide
probe is one of the following probes: SEQ ID NO. 1--atatctacgc
attccaccgc ttcacaagg; SEQ ID NO. 2--atatttacgc attttaccgc
tacacatgg; SEQ ID NO. 3--gccccactcg taagaggcat gatgatttg; SEQ ID
NO. 4--gccctagaca taaggggcat gatgatttg; SEQ ID NO. 5--cgaattgcag
acttcaatcc gaactgaga; SEQ ID NO. 6--cgaattgcag actccaatcc
gaactgaga; SEQ ID NO. 7--cgagttgcag actacaatcc gaactgaga; SEQ ID
NO. 8--cgaattgcag ccctcaatcc gaactgaga; SEQ ID NO. 9--tactactcag
gcggatcatt taatgcgtta g; SEQ ID NO. 10--tactactcag gcggagaact
taatgcgtta t; and SEQ ID NO. 11--tactacccag gcgggatgtt taatgcgtta
g.
[0012] In one aspect of the invention, cell culture supernates or
cultured cell pellet samples are lysed to form samples. Samples are
hybridized with a hybridization solution containing the
oligonucleotide probes having the sequences SEQ ID NOs. 12-15
labeled with a capture ligand and oligonucleotide probes having the
sequences SEQ ID NOs. 1-11 labeled with a component of a detection
system comprising a detection ligand and wherein the probes are
targeted to the 16S ribosomal RNA of at least eight of the most
common mycoplasma species typically found to contaminate cell
cultures, that is: M. hyorhinus, M. arginini, M. fermentans, M.
orale, M. pirum, M. hominis, M. salivarium, and A. laidlawii. In
one embodiment the capture ligand is biotin and the detectable
ligand is digoxigenin. In one method of the invention the
hybridization solution is contacted with a solid phase coated with
a capture receptor chosen to specifically bind to the capture
ligand and the small rRNA probe hybrid is captured. Following a
wash to remove unbound material, another component of the detection
system comprising a detection receptor chosen to specifically bind
to the detection ligand where the detection receptor is bound to a
signal generating label, is contacted with the solid phase for a
time sufficient for the detection ligand to bind to the detection
receptor. A substrate solution that reacts with the signal
generating label to produce a detectable signal is then added and
the detectable signal is produced in proportion to the presence of
mycoplasma rRNA in the original sample. In one embodiment of the
invention the signal generating label produces a color, which color
development may optionally be amplified by adding an amplifier
solution.
DETAILED DESCRIPTION OF THE INVENTION
[0013] This invention relates to a method and kit to detect
mycoplasma which has contaminated cell cultures in a laboratory
setting. The method of the invention includes the use of a first
and a second oligonucleotide probe, each being complementary or
substantially complementary to a portion of a 16S ribosomal RNA of
a mycoplama species. The terms "complementary" and "substantially
complementary," as used herein, refer to sequences that may base
pair hybridize. Complementary nucleotides are, generally, adenine
(a) and thymine (t) or uracil (u), and cytosine (c) and guanine
(g). Two single stranded RNA or DNA molecules are said to be
substantially complementary when the nucleotides of one strand,
optimally aligned and compared and with appropriate nucleotide
insertions or deletions, pair with at least 50% of the nucleotides
of the other strand, preferably at least 60% of the nucleotides,
more preferably at least 70% of the nucleotides, still more
preferably, at least 80% of the nucleotides, and still more
preferably between 90% and 100% of the nucleotides. In one
embodiment of this invention, two or more different first
oligonucleotide probes are contacted with the sample and two or
more different second oligonucleotide probes are contacted with the
sample. In this embodiment, each oligonucleotide probe will be
substantially complementary to a portion of the 16S ribosomal RNA
of a mycoplasma species. In the method of the invention, one second
oligonucleotide probe may be substantially complementary with
approximately 90% of the nucleotides of the probe pairing with the
RNA of one species of mycoplasma present in the sample but may only
be substantially complementary with approximately 75% of the
nucleotides of the probe pairing with the RNA of another species of
mycoplasma present in the sample. However, the presence of both
mycoplasma species will be detected using the method of the assay.
In one aspect of the invention, each of the first oligonucleotide
probes will be substantially complementary with greater than 90% of
the nucleotides of the probe pairing with a portion of the 16S
ribosomal RNA of each of the eight most common mycoplasma species
found to contaminate cultures.
[0014] In some embodiments, a first oligonucleotide probe is
labeled with a capture ligand that is chosen to bind specifically
with a capture receptor bound to a solid phase where the label does
not prohibit the hybridization of the first oligonucleotide probe
to a portion of the 16S ribosomal RNA of a mycoplasma species. As
used herein, the terms "ligand" and "receptor" are used to refer to
a reagent or substance that is a binding pair member that is
capable of recognizing the specific spatial and/or charge
configuration of the other binding pair member and of binding
specifically with it, where a ligand is one member of the binding
pair and a receptor is the other member of the binding pair.
[0015] Binding pairs are well known and include the following:
antigen-antibody and nucleic acid-nucleic acid binding protein,
biotin and avidin, biotin and streptavidin, carbohydrates and
lectins, complementary peptide sequences, effector and receptor
molecules, enzyme cofactors and enzymes, enzyme inhibitors and
enzymes, polymeric acids and bases, and the like. Where the binding
pair member or reagent is described herein as an antibody, the term
"antibody" is intended to encompass an effective portion thereof
retaining specific binding activity for the substance or element.
Effective portions include, for example, Fv, scFv, Fab, Fab.sub.2,
and heavy chain variable regions or a chimeric molecule or
recombinant molecule or an engineered protein comprising any of the
above-mentioned portions.
[0016] In some embodiments of the invention, the capture ligand is
biotin and the capture receptor is streptavidin, avidin or an
anti-biotin antibody.
[0017] As used herein, "bound to" or "coated with" with reference
to the solid phase encompasses all mechanisms for binding
antibodies and proteins and other substances, directly or
indirectly to surfaces of solid phases so that when the solid phase
is contacted with a solution containing the sample the capture
receptor bound to the solid phase remains associated with the
surface. Such mechanisms include chemical or biochemical linkage
via covalent attachment, attachment via specific biological binding
(e.g., biotin/streptavidin), coordinative bonding such as
chelate/metal binding, or the like. In one aspect of the invention,
streptavidin is linked to the solid surface with chemical
attachment.
[0018] "Solid phase" as used herein refers to an insoluble material
to which one component of the detection method may be bound. Known
materials of this type include hydrocarbon polymers such as
polystyrene and polypropylene, glass, metals and gels. Such
supports may be in the form of beads, tubes, strips, disks,
microplates and the like. Polystyrene microplates are desirably
used with the detection method of the system.
[0019] As used herein, "specific binding" and "specifically bound"
means that the reagent, substance or moiety is a binding pair
member that binds or is bound to a desired substance or element
with a higher binding affinity and/or specificity to the substance
or element than to any other moiety present in the sample or used
in the assay method.
[0020] In some embodiments, a second oligonucleotide probe used is
labeled with a first component of a detection system comprising a
detection ligand, that does not prevent the hybridization of the
second oligonucleotide probe to a portion of the 16S ribosomal RNA
of at least one species of mycoplasma. The first component of the
detection system will react with a second component of the
detection system comprising a detection receptor chosen to
specifically bind to the detection ligand and wherein the detection
receptor is labeled with a signal generating moiety. The signal
generating moiety may be a chemical label such as an enzyme, a
fluorescent compound, a radioisotope, a chromophore, or any other
signal generating moiety, provided that when the signal generating
moiety is bound to the detection receptor the detection receptor
retains its capacity to specifically bind to the detection ligand.
"Detection system," and the like, as exemplified below, refers to a
chemical system that generates a detectable signal. In one aspect
of the invention, the detection system includes as a detection
ligand a hapten or protein that may be attached to the second
oligonucleotide probe without interfering with its capacity to
hybridize with a substantially complementary sequence, including
without limitation, digoxigenin. The detection receptor in this
embodiment is an antibody to digoxigenin labeled with a signal
generating moiety that is an enzyme where the detectable signal may
be generated by exposing the labeled reagent to a particular
substrate and incubating for a signal such as color, fluorescence
or luminescence development. In a preferred embodiment, the enzyme
is alkaline phosphatase and the detection systems described in U.S.
Pat. Nos. 4,446,231, 4,595,655, and 4,598,042, the relevant
portions of which are herein incorporated by reference, are used.
Briefly, the detection systems described in those patents discloses
a method where the signal generating moiety is an enzyme which
converts a precursor in a substrate into a cycling vector which in
turn is interconverted in a cycling detection system by contacting
it with a secondary system and the signal generated by the enzyme
is amplified by the enzyme constantly increasing the amount of
cycling factors in the system.
[0021] "Kit" as used herein refers to a combination of reagents
usually formulated with necessary buffers, salts and stabilizers,
where the reagents are premeasured so as to at least substantially
optimize the performance of the detection method.
[0022] One aspect of the invention provides for the use of a test
kit to detect the presence of mycoplasma in a sample. In this
embodiment, the kit includes at least two different first
oligonucleotide probes. In one embodiment, a mycoplasma detection
kit for detecting the presence of mycoplasma contamination
comprises two or more different first oligonucleotide probes
wherein each first oligonucleotide probe is substantially
complementary to a portion of a 16S ribosomal RNA of a mycoplasma
species and is labeled with a capture ligand; two or more different
second oligonucleotide probes wherein the second oligonucleotide
probe is substantially complementary to a different portion of the
16S ribosomal RNA of the mycoplasma species than any of the first
oligonucleotide probes and each is labeled with a first component
of a detection system, wherein the first component comprises a
detection ligand; a solid phase coated with a capture receptor
chosen to specifically bind to the capture ligand on the first
probe; and a detection solution comprising a second component of
the detection system, wherein the second component comprises a
detection receptor labeled with a signal generating moiety. In a
preferred embodiment, the kit comprises first oligonucleotide
probes of each of SEQ ID NOs. 12-15 and second oligonucleotide
probes of each of SEQ ID NOs. 1-11.
[0023] Embodiments of the invention select the first and second
oligonucleotide probes to minimize cross-reactivity with other
bacteria species. Embodiments of the invention also select the
first and second oligonucleotide probes to minimize the formation
of heterodimers of the first and second oligonucleotide probes
which can result in high levels of background noise. Thus the
choice of appropriately matched first and second oligonucleotide
probes is an important aspect of the invention. Embodiments of the
invention comprising one or more first oligonucleotide probes
selected from ID SEQ NOs. 12-15 and one or more second
oligonucleotide probes selected from ID SEQ NOs. 1-11 provide for
appropriate matches of first and second oligonucleotide probes for
detection of mycoplasm contamination by minimizing crossreactivity
with other bacteria species while also minimizing interaction
between the probes.
[0024] In another embodiment of the invention, the signal
generating moiety is an enzyme and the kit further includes a
substrate solution that includes reagents that will react with the
enzyme to generate a detectable signal, and in a further aspect of
the invention the kit further includes an amplifier solution
comprising reagents that will amplify the generation of the
detectable signal being produced.
[0025] The kit of the invention may further include a cell lysis
diluent that includes water treated with diethylpyrocarbonate
(DEPC) at a concentration sufficient to inactivate RNase,
RNase-free Trizma hydrochloride (Tris[hydroxymethyl]aminomethane
hydrochloride), Trizma base (Tris[hydroxymethyl]aminomethane),
calcium chloride dihydrate, and proteinase K.
[0026] The following examples are illustrative of the invention and
is not intended to limit the scope of the invention as set out in
the appended claims.
EXAMPLE 1
The following Example may be performed using the Mycoplasma
Detection Kit, the MycoProbe.RTM. Mycoplasma Detection Kit,
commercially available from R&D Systems, Inc., Minneapolis,
Minn.
Sample Preparation
[0027] Cell lysate supernates (15 .mu.L/well) or cell pellets
(4,700 to 19,000 cells/well) are used as samples in this method.
Cell pellet samples are stored on ice until lysed or stored at
.ltoreq.-20.degree. C. for use at a later time. Cell lysate samples
are prepared using the following procedure: Four hundred
microliters of cell lysis diluent (diluent included water treated
with diethylpyrocarbonate (DEPC) at a concentration sufficient to
inactivate RNase, RNase-free Trizma hydrochloride
(Tris[hydroxymethyl]aminomethane hydrochloride), Trizma base
(Tris[hydroxymethyl]aminomethane), calcium chloride dihydrate, and
proteinase K, hereinafter referred to as "Cell Lysis Diluent") is
added to a cell pellet containing 5.times.10.sup.5 cells to obtain
a final concentration of 1.25.times.10.sup.6 cells per milliliter.
The cells are pipetted up and down several times until resuspended
and vortexed for 15 to 20 seconds. The cell lysate is again diluted
with Cell Lysis Diluent to obtain a final concentration of
approximately 3.times.10.sup.4 to approximately 1.2.times.10.sup.5
cells per milliliter.
[0028] Cell culture supemate samples are similarly prepared. All
cell culture supernate samples are diluted 10-fold in Cell Lysis
Diluent and vortexed for 15-20 seconds.
Assay Procedure
[0029] The hybridization plate (a 96 well plate) is prepared by
washing twice with wash buffer. Excess wash buffer is removed by
decanting or aspirating. The plate is inverted and blotted against
clean paper towels.
[0030] Each well receives 50 microliters of cDNA Probes including
all of the oligonucleotide probes listed in Table 1. These probes
of Table 1 are the first and second oligonucleotide probes of the
invention, wherein each of the first oligonucleotide probes is
labeled with biotin and each of the second oligonucleotide probes
is labeled with the first component of the detection system,
digoxigenin. TABLE-US-00001 TABLE 1 Detection and Capture Probes
SEQ ID Capture/ Tm .epsilon.(nmoles/ .epsilon.(.mu.g/ NO. Detection
Sequence Length (2 + 4) A260) A260) No. 1 Detection atatctacgc
attccaccgc 29 79.8 3.66 32.4 ttcacaagg No. 2 Detection atatttacgc
attttaccgc 29 74.8 3.60 32.1 tacacatgg No. 3 Detection gccccactcg
taagaggcat 29 81.7 3.58 32.2 gatgatttg No. 4 Detection gccctagaca
taaggggcat 29 79.3 3.47 31.5 gatgatttg No. 5 Detection cgaattgcag
acttcaatcc 29 79.2 3.48 31.3 gaactgaga No. 6 Detection cgaattgcag
actccaatcc 29 80.8 3.50 31.3 gaactgaga No. 7 Detection cgagttgcag
actacaatcc 29 77.6 3.44 31.0 gaactgaga No. 8 Detection cgaattgcag
ccctcaatcc 29 83.9 3.57 31.9 gaactgaga No. 9 Detection tactactcag
gcggatcatt 31 77.0 3.32 31.9 taatgcgtta g No. 10 Detection
tactactcag gcqqagaact 31 76.2 3.29 31.7 taatgcgtta t No. 11
Detection tactacocag gcgggatgtt 31 81.2 3.33 32.1 taatgcgtta g No.
12 Capture ggataacgct tgcaacctat 29 76.2 3.57 32.0 gtattaccg No. 13
Capture ggtgtgtaca agacccgaga 30 77.7 3.34 31.1 acgtattcac No. 14
Capture ggtgtgtaca aaacccgaga 30 77.6 3.33 31.0 acgtattcac No. 15
Capture ggtgtgtaca aaccccgaga 30 79.2 3.39 31.4 acgtattcac
[0031] One hundred fifty microliters of a positive control,
negative control or sample, either the cell lysate or cell culture
supemate lysate, are added to each of the designated wells in the
prepared hybridization plate and then covered with a plate sealer.
The positive control used in this example is complementary to one
detection probe and one capture probe. It is intended to provide a
strong positive signal to indicate that the assay is performed
correctly. The sequence for the positive control for use in this
example is SEQ ID NO. 16 caaatcatca tgcctcttac gagtggggcg
tgaatacgtt ctcgggtctt gtacacacc. The negative control contains only
Cell Lysis Diluent and no cellular sample. A float collar is
applied to the hybridization plate and the plate incubated for 60
minutes in an approximately 65 degree C. water bath. A polystyrene
microplate coated with streptavidin (the "Streptavidin Plate") is
washed twice with wash buffer and the excess buffer is removed. One
hundred fifty microliters of each well of the hybridization plate
are then transferred from each well of the hybridization plate to a
designated well of the Streptavidin Plate and a new plate sealer is
applied. The plate is incubated for 60 minutes at room temperature
on a horizontal orbital shaker set at 500.+-.50 rpms. The
Streptavidin Plate is washed four times with wash buffer and then
excess wash buffer is removed. Each well receives 200 .mu.L of
anti-digoxigenin conjugate (21 mL of a polyclonal antibody against
digoxigenin, conjugated to alkaline phosphatase) and the plate is
covered with a new plate sealer. The plate is again incubated for
60 minutes on the shaker at a temperature of approximately
20-25.degree. C. The Streptavidin Plate is washed six times with
wash buffer and then excess wash buffer is removed. 50 .mu.L of
substrate solution (lyophilized NADPH with stabilizers in a
buffered solution) is added to each well and then the plate is
covered with a new plate sealer. The plate is again incubated for
60 minutes on the shaker at a temperature of approximately
20-25.degree. C. The plate is not washed. 50 .mu.L of amplifier
solution (buffered solution containing INT-violet with stabilizers)
is added to each well and the plate covered with a new plate
sealer. The samples are incubated for 30 minutes on the shaker at a
temperature of approximately 20-25.degree. C. and not washed. At
the end of this time, 50 .mu.L of a stop solution (2 N sulfuric
acid) is added to each well. The optical density (OD) of each well
was determined within 30 minutes, using a microplate reader set to
490 nm. If wavelength correction is available, the optical density
is set to 650 nm or 690 nm. If wavelength correction is not
available, readings at 650 nm or 690 nm are subtracted from the
readings at 490 nm to correct for optical imperfections in the
plate. Readings made directly at 490 nm without correction may be
higher and, therefore, less accurate.
[0032] The results are calculated by determining the average of the
duplicate optical density readings for each control and sample. The
average negative control optical density value is subtracted from
all average optical density values. The calculated positive control
optical density value should be greater than or equal to 1.5. The
results from a calculated sample of OD values are determined by
using Table 2, below. Through development and validation, it was
discovered that values less than 0.05 OD are negative. Samples with
values above 0.10 are positive. Values of 0.05-0.10 OD require
retesting after a few days. If after a few days the sample
continues to have the same OD level (0.05-0.10 OD), then it is
negative. However, if the OD level increases, then it is positive.
TABLE-US-00002 TABLE 2 Calculation of Results OD Values
(calculated) Result Interpretation <0.05 Negative No mycoplasma
detected. 0.05-0.10 Inconclusive Sample is suspect for Mycoplasma.
Continue to culture for an additional 2-3 days and repeat the test.
If sample give a similar OD, then no Mycoplasma are detected.
>0.10 Positive Mycoplasma detected.
EXAMPLE 2
[0033] The sensitivity of the method of the invention was shown by
growing each Mycoplasma species in a pure culture serially diluted
and tested with the methods of Example 1. The sensitivity results
are set forth in Table 3, below. TABLE-US-00003 TABLE 3 Sensitivity
Sensitivity Mycoplasma Species (CFU/well) Mycoplasma arginini 15
Mycoplasma orale 65 Mycoplasma fermentans 75 Acholeplasma laidlawii
240 Mycoplasma hyorhinis 560 Mycoplasma pirum 30 Mycoplasma hominis
225 Mycoplasma salivarium 2500 CFU = Colony Forming Units
EXAMPLE 3
Sample Preparation
[0034] Supemate samples of log phase growth CTLL-2 cells, a mouse
cytotoxic T lymphocyte cell line, were tested for possible
mycoplasma contamination. Supemates can be harvested at any time
and stored on ice or frozen at .ltoreq.-20.degree. C. until use.
When ready to assay, dilute the supemate sample 10-fold with cell
lysis diluent (diluent included water treated with
diethylpyrocarbonate (DEPC) at a concentration sufficient to
inactivate RNase, RNase-free Trizma hydrochloride
(Tris[hydroxymethyl]aminomethane hydrochloride), Trizma base
(Tris[hydroxymethyl]aminomethane), calcium chloride dihydrate, and
proteinase K, hereinafter referred to as "Cell Lysis Diluent").
Allow the supemate sample to thaw on ice if the sample has been
stored at .ltoreq.-20.degree. C. For this experiment, 33 .mu.L of
supernate sample was diluted with 297 .mu.L of Cell Lysis Diluent,
vortexed and stored on ice. The sample can also be frozen at
.ltoreq.-20.degree. C, until used. If stored at .ltoreq.-20.degree.
C., sample should be thawed on ice before use.
Assay Procedure
[0035] The hybridization plate (a 96 well plate) was prepared by
washing twice with wash buffer. Excess wash buffer was removed by
decanting or aspirating. The plate was inverted and blotted against
clean paper towels.
[0036] Each well received 50 microliters of cDNA Probes including
all of the oligonucleotide probes listed in Table 1. The probes of
Table 1 are the first and second oligonucleotide probes of the
invention, wherein each of the first oligonucleotide probes was
labeled with biotin and each of the second oligonucleotide probes
was labeled with the first component of the detection system,
digoxigenin.
[0037] One hundred fifty microliters of a positive control,
negative control or sample were added to each designated wells in
the prepared hybridization plate and then covered with a plate
sealer. The positive control used in this example is complementary
to one detection probe and one capture probe. It was intended to
provide a strong positive signal to indicate that the assay was
performed correctly. The sequence for the positive control used in
this example was ID SEQ NO. 16 caaatcatca tgcctcttac gagtggggcg
tgaatacgtt ctcgggtctt gtacacacc. The negative control contained
only Cell Lysis Diluent buffer and no cellular sample. A float
collar was applied to the hybridization plate and the plate
incubated for 60 minutes in an approximately 65 degree C. water
bath. A polystyrene microplate coated with streptavidin (the
"Streptavidin Plate") was washed twice with wash buffer and the
excess buffer was removed. One hundred fifty microliters of each
well of the hybridization plate were then transferred from each
well of the hybridization plate to a designated well of the
Streptavidin Plate and a new plate sealer applied. The plate was
incubated for 60 minutes at room temperature on a horizontal
orbital shaker set at 500.+-.50 rpms. The Streptavidin Plate was
washed four times with wash buffer and then excess wash buffer
removed. Each well received 200 .mu.L of anti-digoxigenin conjugate
(21 mL of a polyclonal antibody against digoxigenin, conjugated to
alkaline phosphatase) and the plate was covered with a new plate
sealer. The plate was again incubated for 60 minutes on the shaker
at a temperature of approximately 20-25.degree. C. The Streptavidin
Plate was washed six times with wash buffer and then excess wash
buffer removed. 50 .mu.L of substrate solution (lyophilized NADPH
with stabilizers in a buffered solution) was added to each well and
then the plate was covered with a new plate sealer. The plate was
again incubated for 60 minutes on the shaker at a temperature of
approximately 20-25.degree. C. The plate was not washed. 50 .mu.L
of amplifier solution (buffered solution containing INT-violet with
stabilizers) was added to each well and the plate covered with a
new plate sealer. The samples were incubated for 30 minutes on the
shaker at a temperature of approximately 20-25.degree. C. and not
washed. At the end of this time, 50 .mu.L of a stop solution (2 N
sulfuric acid) was added to each well. The optical density (OD) of
each well was determined within 30 minutes, using a microplate
reader set to 490 nm. If wavelength correction was available, the
optical density was set to 650 nm or 690 nm. If wavelength
correction was not available, readings at 650 nm or 690 nm were
subtracted from the readings at 490 nm to correct for optical
imperfections in the plate. Readings made directly at 490 nm
without correction may be higher and, therefore, less accurate.
[0038] The results were calculated by determining the average of
the duplicate optical density readings for each control and sample.
The average negative control optical density value was subtracted
from all average optical density values. The calculated positive
control optical density value should be greater than or equal to
1.5. Results are recorded in Table 4.
EXAMPLE 4
Sample Preparation
[0039] A supemate sample of log phase growth BaF3 cells, a mouse
hematopoietic cell line, was tested for possible mycoplasma
contamination. For this experiment, 33 .mu.L of supemate sample was
diluted with 297 .mu.L of Cell Lysis Diluent, vortexed and stored
on ice until assayed.
Assay Procedure
The assay procedure was performed as described above in Example 3.
Results are recorded in Table 4.
EXAMPLE 5
Sample Preparation
[0040] A supemate sample of log phase growth HepG2 cells, a human
hepatocellular carcinoma cell line, was tested for possible
mycoplasma contamination. For this experiment, 33 .mu.L of supemate
sample was diluted with 297 .mu.L of Cell Lysis Diluent, vortexed
and stored on ice until assayed.
Assay Procedure
The assay procedure was performed as described above in Example 3.
Results are recorded in Table 4.
EXAMPLE 6
Sample Preparation
[0041] Log phase growth A431 cells, a human epidermoid carcinoma
cell line, were tested for possible mycoplasma contamination.
5.times.10.sup.5 cells were harvested and pelleted. The cell pellet
was stored on ice until prepared for the assay. Alternatively, the
pellet can be stored at .ltoreq.-20.degree. C. for use at a later
time. If stored at .ltoreq.-20.degree. C., the pellet should be
thawed on ice before prepared for the assay. Cell lysate samples
were prepared using the following procedure: Four hundred
microliters of Cell Lysis Diluent was added to the cell pellet
containing 5.times.10.sup.5 cells to obtain a final concentration
of 1.25.times.10.sup.6 cells per milliliter. The cells were
pipetted up and down several times until resuspended and vortexed
for 15 to 20 seconds. This cell lysate is further diluted 40- to
10-fold with Cell Lysis Diluent to obtain a final concentration of
approximately 3.times.10.sup.4 to approximately 1.2.times.10.sup.5
cells per milliliter. In this experiment, 33 .mu.L of cell lysate
at 1.25.times.10.sup.6 cells/mL was diluted with 297 .mu.L of Cell
Lysis Diluent to obtain a final concentration of
1.25.times.10.sup.5 cells/mL. This sample was stored on ice until
assayed. Alternatively, the cell lysate sample can be stored at
.ltoreq.-20.degree. C. for use in a later assay. If stored at
.ltoreq.-20.degree. C., the cell lysate sample should be thawed on
ice before use in the assay.
Assay Procedure
The assay procedure was performed as described above in Example 3.
Results are recorded in Table 4.
EXAMPLE 7
Sample Preparation
[0042] Log phase growth K562 cells, a human chronic myelogenous
leukemia cell line, were tested for possible mycoplasma
contamination. 5.times.10.sup.5 cells were harvested and pelleted.
The cell pellet was stored on ice until prepared for the assay.
Cell lysate samples were prepared using the following procedure:
Four hundred microliters of Cell Lysis Diluent was added to the
cell pellet containing 5.times.10.sup.5 cells to obtain a final
concentration of 1.25.times.10.sup.6 cells per milliliter. The
cells were pipetted up and down several times until resuspended and
vortexed for 15 to 20 seconds. This cell lysate was further diluted
by adding 297 .mu.L of Cell Lysis Diluent to 33 .mu.L of cell
lysate at 1.25.times.10.sup.6 cells/mL to obtain a final
concentration of 1.25.times.10.sup.5 cells/mL. This sample was
stored on ice until assayed.
Assay Procedure
The assay procedure was performed as described above in Example 3.
Results are recorded in Table 4.
EXAMPLE 8
Sample Preparation
[0043] A second culture of log phase growth K562 cells were tested
for possible mycoplasma contamination. 5.times.10.sup.5 cells were
harvested and pelleted. The cell pellet was stored on ice until
prepared for the assay. Cell lysate samples were prepared using the
following procedure: Four hundred microliters of Cell Lysis Diluent
was added to the cell pellet containing 5.times.10.sup.5 cells to
obtain a final concentration of 1.25.times.10.sup.6 cells per
milliliter. The cells were pipetted up and down several times until
resuspended and vortexed for 15 to 20 seconds. This cell lysate was
further diluted by adding 297 .mu.L of Cell Lysis Diluent to 33
.mu.L of cell lysate at 1.25.times.10.sup.6 cells/mL to obtain a
final concentration of 1.25.times.10.sup.5 cells/mL. This sample
was stored on ice until assayed.
Assay Procedure
[0044] The assay procedure was performed as described above in
Example 3. Results are recorded in Table 4. TABLE-US-00004 TABLE 4
Dilution or OD Values Sample Sample Type Cells/mL (calculated)
Result CTLL-2 Supernate 1:10 1.025 Positive BaF3 Supernate 1:10
0.183 Positive HepG2 Supernate 1:10 0.000 Negative A431 Cell Lysate
1.2 .times. 10.sup.5 1.042 Positive K562 Cell Lysate 1.2 .times.
10.sup.5 1.912 Positive K562 Cell Lysate 1.2 .times. 10.sup.5 0.005
Negative
EXAMPLE 9
[0045] The method and kit of the invention as described in Example
1 were compared to a standard Agar plating method and a PCR method
using a mycoplasma detection kit available from ATCC (American
Tissue Culture Collection, Monassas, VA). As can be seen from the
results set forth in Table 5, the method of the invention,
designated MycoProbe.RTM., identified one sample missed by the PCR
method. TABLE-US-00005 TABLE 5 Mycoplasma Detection Methods
Comparison Mycoplasma MycoProbe .RTM. PCR (ATCC) Agar Plating A.
laidlawii + + + M. arginini + + + M. fermentans + + + M. hominis +
+ + M. hyorhinis + + + M. orale + + + M. pirum + - + M. salivarum +
+ + A = Acholeplasma
EXAMPLE 10
[0046] Assay cross-reactivity of the method and kit described in
Example 1 was tested using 1.times.10.sup.4 CFU/well of microbes
and 15,000 cells/well of mammalian cells listed below in Table 6:
Cross-reactivity. Results were determined using the OD levels
indicated in Table 2. This assay recognized the eight mycoplasma
species previously listed in the Sensitivity Table (Table 2) and
two closely related prokaryote species (based on 16S rRNA
homology), Ureaplasma (U.) urealyticum and Lactobacillus (L.)
casei. U. urealyticum is a mycoplasma associated with human
urogenital diseases and is not found usually as a cell culture
contaminant. U. urealyticum was detectable using as few as
2.7.times.10.sup.3 CFU/well. L. casei is a lactic acid fermenting
bacteria that is not found as a cell culture contaminant. No
significant cross-reactivity was observed for other microbes in the
panel. Mammalian cells did not show cross-reactivity or
interference when tested using the recommended concentration range.
TABLE-US-00006 TABLE 6 Cross-Reactivity Organism Classification
Result OD Ureaplasma urealyticum Mollicute (mycoplasma) Positive
0.168 Lactobacillus casei Gram Positive Bacteria Positive 1.748
Bacillus subtilis Gram Positive Bacteria Negative 0.045 Escherichia
coli Gram Negative Bacteria Negative 0.009 Torulopsis candida Yeast
Negative 0.004 Cryptococcus albidus Yeast Negative 0.001 Geotrichum
sp. Mold Negative 0.002 Penicillium sp. Mold Negative 0.002
Cladosporium sp. Mold Negative 0.002 Human K562 Negative 0.014
Mouse EL-4 Negative 0.000 Rat NR-8383 Negative 0.000
[0047]
Sequence CWU 1
1
16 1 29 DNA Artificial Sequence is synthesized 1 atatctacgc
attccaccgc ttcacaagg 29 2 29 DNA Artificial Sequence is synthesized
2 atatttacgc attttaccgc tacacatgg 29 3 29 DNA Artificial Sequence
is synthesized 3 gccccactcg taagaggcat gatgatttg 29 4 29 DNA
Artificial Sequence is synthesized 4 gccctagaca taaggggcat
gatgatttg 29 5 29 DNA Artificial Sequence is synthesized 5
cgaattgcag acttcaatcc gaactgaga 29 6 29 DNA Artificial Sequence is
synthesized 6 cgaattgcag actccaatcc gaactgaga 29 7 29 DNA
Artificial Sequence is synthesized 7 cgagttgcag actacaatcc
gaactgaga 29 8 29 DNA Artificial Sequence is synthesized 8
cgaattgcag ccctcaatcc gaactgaga 29 9 31 DNA Artificial Sequence is
synthesized 9 tactactcag gcggatcatt taatgcgtta g 31 10 31 DNA
Artificial Sequence is synthesized 10 tactactcag gcggagaact
taatgcgtta t 31 11 31 DNA Artificial Sequence is synthesized 11
tactacccag gcgggatgtt taatgcgtta g 31 12 29 DNA Artificial Sequence
is synthesized 12 ggataacgct tgcaacctat gtattaccg 29 13 30 DNA
Artificial Sequence is synthesized 13 ggtgtgtaca agacccgaga
acgtattcac 30 14 30 DNA Artificial Sequence is synthesized 14
ggtgtgtaca aaacccgaga acgtattcac 30 15 30 DNA Artificial Sequence
is synthesized 15 ggtgtgtaca aaccccgaga acgtattcac 30 16 59 DNA
Artificial Sequence is synthesized 16 caaatcatca tgcctcttac
gagtggggcg tgaatacgtt ctcgggtctt gtacacacc 59
* * * * *